It has recently become apparent that the human VH locus is highly heterogeneous. As such, it is possible that individual differences in the coding region content of the VH germline repertoire may underlie important differences in antibody response, and predisposition to the autoimmune diseases SLE and RA. This proposal will elucidate the immunological consequences of VH gene heterogeneity in two distinct systems. First, sequence-specific oligonucleotides will be employed to identify VH germline elements, and to distinguish among their highly homologous variants. The analysis will initially be directed toward known VH genes, including VH elements which encode autoantibodies, VH elements which are and which are not found in the fetally expressed repertoire, and VH elements which map to dispersed locations within the VH locus. The nucleotide sequence of detected VH elements, and of their suspected allelic variants, will be determined by PCR amplification of genomic DNA, and cloning into M13. A panel of oligonucleotide probes developed in the preceding phase of the project will be employed to determine prevalences of the identified VH genes, and of their combinations, in 80 normal individuals. The prevalence of several candidate and marker VH genes, selected on the basis of informative polymorphism or suspected allelic relationships, will be measured in 50 patients with SLE and 50 patients with RA. In an affected sibling study, segregation of VH haplotypes will be determined in 10 families containing sibling pairs with SLE, and 20 with RA. HLA-DR type will be determined by hybridization analysis of the entire study population. This proposal will also establish a system for study of disease-related VH gene expression by examining the genetic basis for Ab binding to a model Ag, Staphylococcal Protein A (SPA). Specificity for SPA is highly restricted to Ab encoded by the large VH3 family. The mRNA of human B cells polyclonally activated by SPA will be studied by northern hybridization with cloned and oligonucleotide probes to determine VH family and single gene expression in pre- vs. post-activated cells. A filter paper colony blot system will be developed to permit PCR amplification from single B cell clones to analyze VH gene use by Ab which do and do not bind to SPA.